The Experimental Study Of Pancreatic Cancer Using Molecular Probe Targeting MUC5AC

Part ⅠThe preparation and characterization of MR Molecular ProbeTargeted MUC5AC In Pancreatic CancerObjective:To construct PEG modified SPIONs and a novel magnetic molecular probe targeting MUC5AC in pancreatic cancer cells.The characterization of the PEG·SPIONs was discussed in this section.Materials and methods:1.Preparation of PEG·SPIONs:2 mmol of Fe(acac)3,6 g of Polyethylene Glycol(PEG)and 25 mL of Triethylene glycol(TEG)were mixed and purged with nitrogen for 30 min.The reaction mixture was magnetically stirred at 100℃ until all of the reagents were completely dissolved into the solvent.The mixture was heated to 210℃ for 2 h and then refluxed(287℃)for another hour.After cooling to room temperature,the PEG-SPIONs were purified with sodium citrate and ethanol for three times,and dried at room temperature.The purified PEG-SPIONs were weighed and redispersed in borate buffer solution(pH 8.2)for future studies.2.Preparation of PEG · SPIONs:1)16.5uM PEG SPIONs dispersed in Buffer solution(200uL,pH=8.2),and then add anti-MUC5AC(15ug);2)8.0mgEDC/NHS(1-ethyl-3-(3-dimethyl-aminopropyl)-1-carbo-DiImidehydrochlori-d e/N-hydroxysuccinimide)dissolved in deionized water(500uL),12ul was added to the above solution;3)the mixture was shaken for 2h in a swing bed at room temperature.Using a high-speed centrifuge(1X104r/min,15min),the mixture was finally resuspended in preservation solution and 4 ℃ stored.3.Construction of FITC-anti-MUC5AC-PEG·SPIONs:0.6mg of FluoresceinIsothiocyanate(FITC)dissolved in DMSO and then added to 7.11uM of the anti-MUC5AC-PEG SPIONs;after 24h mechanical rotation,the mixture was dialyzed in deionized water(interval 4 hours)to get FITC-anti-MUC5AC-PEG · SPIONs.The whole process must be strictly protected from light4.The morphology and core size of PEG were characterized by Transmission electron microscopy(TEM).Fourier transform infrared spectroscopy(FTIR)was used to detect the groups on the surface of PEG·SPIONs.The magnetic property was performed with X-ray electron diffraction(XRD),a Vibrating Sample Magnetometer(VSM)and a MR imager.The optical properties of FITC-anti-MUC5AC-PEG SPIONs were analyzed by Fluorescence spectroscopy.Results:The PEG-SPIONs had a suitable size(12 nm sized core)and perfect monodisperse.XRD show that PEG SPIONs have three strong diffraction peaks and consistent with standard Fe304 PDF card(JCPDS 11-0614).The magnetic analysis shows the saturation magnetization was 29.9 emu/g and suitable for MR imaging contrast agents.Fourier transform infrared(FTIR)show that PEG was successfully coated on Fe304 surface.The emission peak of FITC-anti-MUC5AC-PEG-SPIONs was around 520nm and emerged a bright yellow-green fluorescence.Conclusions:The molecular probe prepared in this article have good colloidal stability,dispersion and magnetic property and a promising for the further experiment.Part ⅡThe Pancreatic Cancer Cells Cultured With the MUC5AC Molecular Probe in VitroObjective:The pancreatic cancer cell line SW1990 and mouse fibroblast cell line 3T3 cultured with anti-MUC5 AC-PEG-SPIONs was evaluated by pharmacokinetic trials.We research the imaging capabilities and targeting capabilities of the MUC5AC probe in vitro.Materiarls and methods:1.MTT(Microculture tetrazolium assay,MTT)assay was utilized as cell viability evaluation to detect the biomaterial toxicity of cells using the mouse fibroblast cell line as a contrast group.2.PEG-SPIONs、Anti-MUC5AC-PEG·SPIONs were cultured with the pancreatic cells SW1990 for 24h.A Prussian blue staining assay was used to research the cellular uptake of nanoparticles.The iron concentration of each sample was determined by inductively coupled plasma atomic emission spectroscopy(ICP-AES).3.Confocal analysis:FITC-PEG-SPIONs,FITC-anti-MUC5AC-PEG-SPIONs were cultured with the pancreatic cells SW1990 for 15min and then selected images.4.In Vivo Biodistribution and Histology Analysis:The PEG-SPIONs were injected intravenously in Kunming mice(n=3)at a concentration of 2.5 mg/kg body weight.Three mice without injection were used as the blank control.Major organs were removed from mice post-injected with PEG-SPIONs at different intervals.The removed organs were pulverized and treated with nitric acid.The organ/nitric acid solutions were heated at 90℃ for eight hours and then filtered for ICP analysis.Tissue samples were(heart,liver,spleen,lung,kidney and intestine)harvested from mice injected with PEG-SPIONs 15 days post-injection and from those receiving no injection.The histological sections were observed under an optical microscope.Result:These data of MTT show that the nanoparticles PEG-SPIONs manifest lower cytotoxicity.Many blue particles in cells could be seen in cells incubated with anti-MUC5AC-PEG·SPIONs,while the blue particles nearly can’t be seen in cells labeled with PEG·SPIONs.According to the result of confocal microscopy,the pancreatic cancer cells incubated with FITC-anti-MUC5AC-PEG SPIONs have more fluorescence distribution in cytoplasm than the group of FITC-PEG·SPIONs.This demonstrates that the targeted probe can transfect the SW1990 cell line well.The higher uptake amount was anti-MUC5AC-PEG SPIONs compared to the group of PEG-SPIONs.ICP analysis shows PEG SPIONs uptake primarily in the liver,spleen,and intestine.There are no apparent histopathological abnormalities or lesions in major organs compared to those of the control group.Conclusions:FITC-Anti-MUC5AC-PEG·SPIONs with low toxicity can transfect SW1990 cells efficiently.Confocal and Prussian blue staining results show that the targeted probe can enter cells and located in cytoplasm.Therefore,the probe prepared in this experiment FITC-anti-MUC5AC-PEG-SPIONs may be a good molecular probe specific targeting pancreatic tumors.